Developing neurons assemble their functional circuitry in response to intrinsic genetic instructions and complex external clues in the local environment. Some of these environmental dues may come from classical neurotransmitters, such as GABA, produced by early maturing, pioneer neurons. The overall goal of this project is to provide a developmental analysis of GABA, glutamate and melatonin systems in retina. Specifically, we will examine how developmental events regulate the expression of transmitter phenotype and in return, how neurotransmitters influence developmental events. Since these are highly complex problems, we have chosen to study a preparation with relatively simplistic circuitry involving only two major cell types, cone photoreceptors and type A horizontal cells. We have been able to analyze the development of these synapses which are presumably reciprocal between GABAergic horizontal cells and glutamatergic cones, in relative isolation from other maturing cell types during the initial stages of synaptogenesis in the outer plexiform layer of rabbit retina. One of our most important findings has been that blockage of GABA A receptors by picrotoxin at birth either in vivo or in vitro, blocks cone synaptogenesis and leads to disruption of the cone mosaic. This data suggests that GABA produced from pioneering horizontal cells, may be necessary for maintaining the position of cones within a normal mosaic array. These studies will be pursued using a highly multidisciplinary approach which involves previously established microscopy techniques and neurochemical analyses, as well as procedures new to the P.I. such as tissue culture and single cell injections of Lucifer Yellow. 1) We will establish the most suitable preparation using isolated retinas or retinal cultures for the subsequent examination of the cellular and molecular mechanisms involved in the picrotoxin effect. II) If the effect of picrotoxin is linked to its antagonism of endogenous GABA interaction with postsynaptic receptors, then GABA receptors should be present in the outer retina at birth and horizontal cells should release sufficient GABA to activate them. We will test these assumptions first, by localizing GABA receptors with antibodies and second, by measuring endogenous GABA and glutamate release with GCMS under basal conditions and after stimulation with GABA and glutamate analogues. III) A more complete understanding of neurochemical mechanisms will require a detailed appreciation of the anatomical substrate upon these interactions are carried out. Various staining techniques including intracellular injections of Lucifer Yellow, will be used to map the distribution of cone and horizontal cells and to detail the synaptic convergence of these cell types in the outer plexiform layer. IV) In the final set of experiments, we will examine whether or not the melatonin system found in adult photoreceptor cell terminals, is present early in postnatal retina coincident with expression of the glutamate immunoreactivity. If so, additional studies will examine how melatonin might interact with GABA and/or glutamate in influencing retinal development. The results of these experiments should enhance our understanding of 1) synaptogenesis in retinal cell types which are pivotal in establishing the overall visual mosaic; 2) the role of GABA in development and 3) basic aspects of synaptogenesis and neuronal development.